In eukaryotic cells, chromosomal ends are capped by telomeres, which are long tandem-repeat sequences complexed with proteins (Blackburn, E. H., et al., Nat. Med. 12:1133-8 (2006); Szostak, J. W., et al., Cell 29:245-55 (1982)). Telomeres maintain the integrity and stability of chromosomes, which would otherwise undergo incomplete replication, fusion or degradation with each cell division (Cech, T. R., Cell 116:273-9 (2004); Collins, K., Nat. Rev. Mol. Cell. Biol. 7:484-94 (2006)). Telomerase is responsible for telomere elongation and maintenance of chromosomal ends in most eukaryotes (Blackburn, E. H., et al., Nat. Med. 12:1133-8 (2006); Greider, C. W., et al., Cell 43:405-13 (1985)). It has long been known that telomerase is fundamental to cell survival, growth and death (Blasco, M. A., Nat. Chem. Biol. 3:640-9 (2007)). Telomere shortening is associated with ageing and telomerase malfunction is often associated with disease. For instance, most cancer cells have an unusually high level of telomerase activity (Kim, N. W., et al., Science 266:2011-5 (1994)). On the other hand, mutations in telomerase components have been linked to several degenerative diseases such as dyskeratosis congenita and aplastic anemia (Blasco, M. A., Nat. Chem. Biol. 3:640-9 (2007)). Thus, to understand the molecular mechanisms of these diseases and to identify new treatments, it is desirable to regulate telomerase activity in vivo.
Telomerase is a ribonucleoprotein (RNP) complex (Greider, C. W., et al., Nature 337:331-7 (1989)) that consists of one noncoding RNA (known as TERC or TR in humans and TLC1 in Saccharomyces cerevisiae) (Singer, M. S., et al., Science 266:404-9 (1994)) and several proteins, including a reverse transcriptase (TERT in humans and Est2p in Saccharomyces cerevisiae) (Lingner, J., et al., Science 276:561-7 (1997)). The telomerase non-coding RNA not only folds into a structure that tethers proteins but also serves as a template for reverse transcription (Zappulla, D. C., et al., Proc. Natl. Acad. Sci. USA 101:10024-9 (2004)), which leads to the addition of a specific repeated sequence to the chromosome ends. S. cerevisiae TLC1 and its homologs in other organisms (including mammals) have been extensively studied. Several other possible functions (including catalysis) of telomerase RNA have been proposed (Miller, M. C., et al., Proc. Natl. Acad. Sci. USA 99:6585-90 (2002); Qiao, F., et al., Nat. Struct. Mol. Biol. 15:634-40 (2008)). Furthermore, NMR studies and computational modeling coupled with functional analysis have revealed a conserved triple-helix structure within the pseudoknot region of human and K. lactis telomerase RNAs (Shefer, K., et al., Mol. Cell. Biol. 27:2130-43 (2007); Theimer, C. A., et al., Mol. Cell. 17:671-82 (2005)). Recently, Qiao, et al. has presented experimental evidence for the presence of a similar triple-helix structure in yeast TLC1 RNA (Qiao, F., et al., Nat. Struct. Mol. Biol. 15:634-40 (2008)) (FIG. 1A). Changes of 2′-OH groups of nucleotides in and adjacent to the triple-helix region to 2′-H or 2′-OMe (2′-O-methylated) lead to reduction of telomerase activity in yeast and mammalian in vitro systems (Qiao, F., et al., Nat. Struct. Mol. Biol. 15:634-40 (2008)).
Box C/D ribonucleoproteins (RNPs) are modifying enzymes that introduce 2′-O-methylation into rRNAs and snRNAs at specific sites (Yu, Y. T., et al., in H. Grosjean (Ed.): Fine-Tuning of RNA Functions by Modification and Editing, vol. 12, Springer-Verlag, Berlin Heidelberg (2005)). Box C/D RNPs comprise one small RNA (box C/D RNA) and four core proteins (Fibrillarin or Nop1p in S. cerevisiae, 15.5-kDa protein, Nop56 and Nop58) (Yu, Y. T., et al., in H. Grosjean (Ed.): Fine-Tuning of RNA Functions by Modification and Editing, vol. 12, Springer-Verlag, Berlin Heidelberg (2005)). A typical box C/D RNA folds into a unique secondary structure, leaving two short sequences—one between box C and box D′ and one between box C′ and box D—unpaired or single stranded (FIG. 1B). These single-stranded sequences function as guides that base-pair with the natural rRNA and snRNA substrates, thereby directing 2′-O-methylation at specific sites (Bachellerie, J. P., et al., Trends Biochem. Sci. 20:261-4 (1995); Cavaille, J., M. et al., Nature 383:732-5 (1996); Kiss-Laszlo, Z., et al., Cell 85:1077-88 (1996)). Without exception, 2′-O-methylation occurs at the target nucleotide in the substrate RNA that is base-paired to the nucleotide in snoRNA precisely 5 nucleotides upstream from box D (or D′; FIG. 1B) (Cavaille, J., M. et al., Nature 383:732-5 (1996); Kiss-Laszlo, Z., et al., Cell 85:1077-88 (1996)). Once the box C/D snoRNA finds its nucleotide target, fibrillarin, a methyl transferase associated with the box C/D guide RNA, delivers the methyl group to the target nucleotide at the 2′-O position. The “Box D+5 rule” for predicting the site of 2′-O-methylation guided by box C/D RNAs has been verified in various organisms including yeast, Xenopus and human, suggesting that RNA-guided 2′-O-methylation of rRNA and snRNA is universal among eukaryotes (Kiss, T. et al., Embo J 20:3617-22 (2001); Kiss, T., et al., Cell 109:145-8 (2001); Peculis, B., Curr. Biol. 7:R480-2 (1997); Smith, C. M., et al., Cell 89:669-72 (1997)). Given the detailed mechanism of RNA-guided RNA 2′-O-methylation, it is possible to design artificial box C/D RNAs to target telomerase RNA in and adjacent to the triple-helix region, thus offering an opportunity to manipulate telomerase activity in vivo.
As disclosed in detail herein, the present inventors show that artificial box C/D RNAs can target 2′-O-methylation at specific sites in and adjacent to the triple-helix structure of telomerase, thereby affecting telomerase activity in vivo. 2′-O-methylation did not affect the steady-state level of TLC1, and 2′-O-methylated TLC1 was incorporated into telomerase RNP. Thus, these results indicate that telomerase activity can be manipulated in vivo.